High performance liquid chromatography (HPLC) of proteins is developing into a major new analytical methodology for the analysis of proteins. The research in this proposal has as its long term objective the pursuit of frontiers in column development, development of necessary theory, and application of the technique of HPLC to polypeptide separations in the microscale, analytical and preparative modes. The significance of this research is that new methods for protein fractionation will be found, the rate of conventional separations will be increased 10 fold or more and the efficiency of doing life science research and development involving polypeptide separations will be improved. The short term objectives of this research are to: 1) develop new column packing materials for size exclusion (SEC), ion exchange (IEC), and reversed phase chromatography (RPC) of proteins and peptides, 2) develop performance "bench marks" for these new columns and existing commercial columns, 3) develop a more quantitative treatment of the chromatography of macromolecules in terms of loading capacity, pore diameter, the influence of surface area and ligand density, matrix contributions to retention and description of the retention process itself and 4) apply the above findings in the preparation of preparative, analytical and macrocolumns.